The aggregation of peptides and proteins into highly organized aggregates such as amyloid fibrils is a very important and fundamental process in biology. Most proteins suffer some degree of aggregation after they are synthesized in the cell, and a variety of cellular mechanisms have evolved to deal with the consequences of aggregation, which range from rendering protein synthesis inefficient to creating toxic species. In some cases amyloid fibril formation is beneficial and necessary for a particular biological function, such as the bacterial adhesins that support biofilm growth, and the secretory storage granules used by mammals for storage and release of protein hormones like insulin. In many other cases amyloid fibril formation is detrimental, being associated with serious human diseases as well as with normal aging. Protein aggregation also plays important roles in biotechnology, in the recombinant synthesis, purification and formulation of protein therapeutic agents. For all of these reasons it is important to come to a better mechanistic understanding of the protein aggregation process, and in particular the events that initiate aggregation, a process termed nucleation. Many cases of spontaneous amyloid fibril formation involve highly complex nucleation mechanisms with multiple molecular species in high flux, greatly complicating nucleation analysis. In other cases, however, nucleation is more simple, and in fact more resembles the classical model of nucleated growth polymerization from basic polymer chemistry. This grant application proposes a series of experiments to unravel the nucleation mechanism of a series of modified polyglutamine (polyQ) molecules, which are an important place to begin to study nucleation because they fall into the second, more simple class of nucleation mechanisms. The basic approach will be to design polyQ sequences containing various mutations that are predicted to have defined conformational effects on the solution structure of the protein, and to carry out detailed nucleation kinetics analyses on these proteins. These analyses will yield important parameters such as the thermodynamic driving force for nucleus formation, and the thermodynamics associated with simple fibril growth (elongation). These experimentally determined values for the energetics of amyloid nucleation and growth will then be compared with expectations based on the known conformational tendencies introduced into the test polyQ molecules by the added mutations. In this way, certain hypotheses for the structural nature of the aggregation nucleus will be tested. Success in this project will provide important clues as to how amyloid formation is nucleated that should have implications over broad areas of biology, such as biological systems affected by amyloid diseases, as well as related protein aggregation processes that occur in normal cell biology and in normal aging. Since it is likely that conformational rearrangements are important even in the more complex type of amyloid nucleation reactions, the results may open the way to new approaches to the study of the more complex and currently largely impenetrable class of nucleation mechanisms. PUBLIC HEALTH RELEVANCE: Proteins are the workhorses of the cell and are the agents by which the blueprints encoded in an organisms DNA are carried out. But proteins, as polymers, have a biophysical tendency to aggregate that is often in competition with the evolved function of the protein to fold into an active enzyme, hormone, etc. In some cases the aggregation ability of certain proteins has been utilized by nature in a positive way to enhance function. But aggregation is most of the time detrimental, being an aspect of normal aging, and a recurring feature of a number of important neurodegenerative diseases and other diseases. Aggregation also causes major problems in biotechnology. This project is designed to study in detail the mechanisms by which protein aggregation is initiated, including a possible intimate relationship to the mechanism of normal protein folding. The results will have wide impact for the understanding of normal and abnormal biology as well as the manufacturing and formulation of protein pharmaceuticals.